PROJECT SUMMARY Neuropathic pain originates from nerve injury and impairs the quality of life for a substantial number of Americans. Nerve injury causes cellular and molecular changes in the spinal cord, but the precise effects on pain-related spinal circuits and cell types has not fully been resolved. Molecularly distinct cell types in a complex tissue can now be comprehensively identified and studied using single cell RNA sequencing (scRNAseq) technologies. In preliminary studies, we found that the scRNAseq approach known as Drop-seq can be used to identify the principle cell types of the mouse spinal cord, including neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells. In this proposal, we hypothesize that single cell sequencing can be used to comprehensively identify cellular and molecular changes that take place in the spinal cord following peripheral nerve injury. To accomplish this, we will complete the following aims: (1) Test the hypothesis that Drop-seq can identify changes in spinal cell populations in a mouse neuropathic pain model, which features prolonged (>21 d) mechanical allodynia?a core behavioral phenotype of neuropathic pain. (2) Test the hypothesis that scRNAseq at varying timepoints can identify cell-specific transitions from acute to chronic pain. Our research will provide the first comprehensive understanding of how spinal cell types, and the neural circuits they make up, change in response to nerve injury?an insult that produces stable behavioral changes indicative of neuropathic pain.